The first vaccines consisted of live, attenuated pathogens. The attenuated forms were either naturally occurring closely related organisms or obtained through serial passages in culture. For example, tuberculosis (TB) in man has for many years been combated by vaccination with an attenuated strain of Mycobacterium bovis, the M. bovis BCG vaccine developed more than 80 years ago. However, although more than 3 billion doses of BCG have been administered (more than any other vaccine) (Bloom and Fine, 1994), it does not always provide satisfactory resistance to humans TB in every population.
Today, a more up-to-date approach is to use highly purified substances, e.g. purified recombinant proteins. These vaccines are well-defined and side-reactions are minimized. Unfortunately, many highly purified substances are not very immunogenic and do not induce a sufficient immune response to confer protection. To do this, the antigen needs some help from immune response potentiating agents called adjuvants. Depending on the pathogen, protection may require that either a humoral or a cell-mediated response predominate. The development of a specific kind of immune response (humoral or cell-mediated) can be determined by the choice of adjuvant.
Protective immunity against an intracellular pathogen like M. tuberculosis requires a cell-mediated immune response, and a suitable adjuvant for a subunit vaccine directed against TB should enhance a Th1 response (Lindblad et al, 1997). It is generally believed that antibodies do not play an important role in immunity to TB whereas cell-mediated release of IFN-gamma (interferon gamma) is the most important cytokine involved in protection (Collins & Kaufmann, 2001).
A large number of adjuvants exist but most of these suffer from numerous problems that preclude their use in humans. The only adjuvants accepted for human use are aluminum-based adjuvants (AlOH-salts) and MF-59, but they both induce Th2-biased responses, which makes them unsuitable for a TB vaccine (Lindblad et al, 1997).
During the past 20-30 years a number of new adjuvant systems have been identified. One example is QS-21, which is a highly purified compound isolated from the bark of the South American tree Quillaja saponaria. QS-21 is a potent adjuvant with low toxicity (Kensil et al, 1991). Lack of ease of production and a high price may be an important issue for QS-21 and other novel, promising adjuvant compounds. Despite the fact that many adjuvant systems have been developed, the need for new adjuvant systems is still recognized (Moingeon et al, 2001) and is evident in the paucity of choices available for clinical use.
Various compounds from mycobacteria have been reported to be immunepotentiating. When lipids extracted from M. bovis BCG were used as adjuvant, a skin test response to ovalbumin was obtained in guinea pigs (Hiu, 1975). Liposomes formed at elevated temperatures from total polar lipids of M. bovis BCG are able to generate a humoral response to ovalbumin, and a vaccine prepared from these polar lipids gave protection in mice upon challenge with tumor cells (WO 03/011336). The effect of total lipids from M. tuberculosis H37Rv as antigen in an experimental TB vaccine for guinea pigs was investigated by Dascher et al (2003). In this study, liposomes based on cholesterol and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) were mixed with a M. tuberculosis H37Rv total lipid extract. After removing the solvent, the lipids were reconstituted with DDA as an adjuvant in PBS buffer. Guinea pigs immunised with this vaccine did not show significant reduction in bacteria, suggesting that this formulation of liposomes mixed with DDA lack a strong antigen or that the formulation of mycobacterial lipids with Cholesterol:DSPC prevent the adjuvenating effect of DDA.
Various purified components from mycobacteria have also been investigated for their adjuvant activity. Purified protein derivative (PPD) did not induce delayed type hypersensitivity reaction on its own, but when Wax D (a mycobacterial cell wall peptidoglycan fragment-arabinogalactan-mycolic acid complex) was added as adjuvant, a strong reaction was observed (Yamazaki, 1969). The immunomodulator SSM or Z-100, a lipid arabinomannan extracted from M. tuberculosis, has antitumor activity (Suzuki et al, 1986). Another mycobacterial cell-derived compound is trehalose 6,6′-dimycolate (cord factor; a mycolic acid containing glycolipid) (Saito et al, 1976). Also, trehalose 6,6′-dimycolate (or synthetic analogues) has immunostimulatory effects and has been included in various adjuvant formulations (McBride et al, 1998; Koike et al, 1998). Taken together, several immunostimulating lipid compounds have been isolated from mycobacteria, but the laborious and thereby expensive purification schemes required makes them unlikely to be included in a future TB vaccine.
Adjuvants exist in many different forms, some of which are surfactant-like and form liposomes, which are vesicles made up of lipid bilayers. The liposomes act as carriers of the antigen (either within the vesicles or attached onto the surface) and may form a depot at the site of inoculation allowing slow, continuous release of antigen. For some time after injection and phagocytosis, liposomal presentation ensures that a specific amount of antigen is made available to single antigen-presenting cells (Glück, 1995). The adjuvant activity of liposomes applies to, a large variety of pathogens (Gregoriadis et al, 2000), and more recently prominent anti-tumor responses characterized by cytotoxic CD8 T cell responses were elicited with therapeutic vaccines adjuvanted with cationic lipids (Siders et al, 2002).
Dimethyldioctadecylammonium-bromide, -chloride, -phosphate, -acetate or other organic or inorganic salts (DDA) is a lipophilic quaternary ammonium compound, which forms cationic liposomes in aqueous solutions at temperatures above 40° C. It promotes cell mediated immunity (Hilgers & Snippe, 1992). Combinations of DDA and other immunomodulating agents have been described. Administration of Arquad 2HT, which comprises DDA, in humans were promising and did not induce apparent side effects (Stanfield, 1973). An experimental vaccine based on culture filtrate proteins from M. tuberculosis and DDA generated a protective immune response against TB in mice (Andersen, 1994). Vaccination of mice with a fusion protein of M. tuberculosis proteins ESAT-6 and Ag85B, and DDA/MPL as adjuvant, provides protection similar to that obtained by BCG vaccination (Olsen et al, 2001). These studies demonstrate that, in contrast to e.g. alum, DDA-based adjuvants are able to induce a protective immune response against TB in mice. Moreover, DDA has been used as an adjuvant for a DNA vaccine against pseudorabies virus leading to enhanced T-cell responses and anti-viral immunity (van Rooij et al, 2002). DDA is therefore a promising choice for development of an adjuvant system for a vaccine against TB and other intracellular pathogens.
As indicated above, new adjuvant systems are clearly required. The ideal adjuvant system, which is the subject matter of the present invention, is cheap and easy to produce, it generates a long-lasting protective, immune response of the right type (Th1 or Th2 depending on the pathogen), it does not elicit unacceptable local reactions, it offers long-term stable presentation of the antigen (depot effect) and it helps to target immune cells.